Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using Infinium HumanMethylation 450 BeadChips

Hepatocellular carcinoma (HCC) incidence has increased in the US and also has one of the fastest growing death rates of any cancer. The purpose of the current study was to discover novel genome-wide aberrant DNA methylation patterns in HCC tumors that are predominantly HCV-related. Infinium HumanMethylation 450K BeadChip arrays were used to examine genome-wide DNA methylation profiles in 66 pairs of HCC tumor and adjacent non-tumor tissues. After Bonferroni adjustment, a total of 130,512 CpG sites significantly differed in methylation level in tumor compared with non-tumor tissues, with 28,017 CpG sites hypermethylated and 102,495 hypomethylated in tumor tissues. Absolute tumor/non-tumor methylation differences ≥ 20% were found in 24.9% of the hypermethylated and 43.1% of the hypomethylated CpG sites; almost 10,000 CpG sites have ≥ 30% DNA methylation differences. Most (60.1%) significantly hypermethylated CpG sites are located in CpG islands, with 21.6% in CpG shores and 3.6% in shelves. In contrast, only a small proportion (8.2%) of significantly hypomethylated CpG sites are situated in islands, while most are found in open sea (60.2%), shore (17.3%) or shelf (14.3%) regions. A total of 2,568 significant CpG sites (2,441 hypermethylated and 127 hypomethylated) covering 589 genes are located within 684 differentially methylated regions defined as regions with at least two significant CpG sites displaying > 20% methylation differences in the same direction within 250-bp. The top 500 significant CpG sites can significantly distinguish HCC tumor from adjacent tissues with one misclassification. Within adjacent non-tumor tissues, we also identified 75 CpG sites significantly associated with gender, 228 with HCV infection, 17,207 with cirrhosis, and 56 with both HCV infection and cirrhosis after multiple comparisons adjustment. Aberrant DNA methylation profiles across the genome were identified in tumor tissues from US HCC cases that are predominantly related to HCV infection. These results demonstrate the significance of aberrant DNA methylation in HCC tumorigenesis.     

The Dynamics of DNA Methylation Covariation Patterns in Carcinogenesis

Recently it has been observed that cancer tissue is characterised by an increased variability in DNA methylation patterns. However, how the correlative patterns in genome-wide DNA methylation change during the carcinogenic progress has not yet been explored. Here we study genome-wide inter-CpG correlations in DNA methylation, in addition to single site variability, during cervical carcinogenesis. We demonstrate how the study of changes in DNA methylation covariation patterns across normal, intra-epithelial neoplasia and invasive cancer allows the identification of CpG sites that indicate the risk of neoplastic transformation in stages prior to neoplasia. Importantly, we show that the covariation in DNA methylation at these risk CpG loci is maximal immediately prior to the onset of cancer, supporting the view that high epigenetic diversity in normal cells increases the risk of cancer. Consistent with this, we observe that invasive cancers exhibit increased covariation in DNA methylation at the risk CpG sites relative to normal tissue, but lower levels relative to pre-cancerous lesions. We further show that the identified risk CpG sites undergo preferential DNA methylation changes in relation to human papilloma virus infection and age. Results are validated in independent data including prospectively collected samples prior to neoplastic transformation. Our data are consistent with a phase transition model of carcinogenesis, in which epigenetic diversity is maximal prior to the onset of cancer. The model and algorithm proposed here may allow, in future, network biomarkers predicting the risk of neoplastic transformation to be identified.     

Non-Steroidal Anti-Inflammatory Drug Use and Genomic DNA Methylation in Blood

Background

Non-steroidal anti-inflammatory drug (NSAID) use is associated with decreased risk of some cancers. NSAID use modulates the epigenetic profile of normal colonic epithelium and may reduce risk of colon cancer through this pathway; however, the effect of NSAID use on the DNA methylation profile of other tissues including whole blood has not yet been examined.

Findings

Using the Sister Study cohort, we examined the association between NSAID usage and whole genome methylation patterns in blood DNA. Blood DNA methylation status across 27,589 CpG sites was evaluated for 871 women using the Illumina Infinium HumanMethylation27 Beadchip, and in a non-overlapping replication sample of 187 women at 485,512 CpG sites using the Infinium HumanMethylation450 Beadchip. We identified a number of CpG sites that were differentially methylated in regular, long-term users of NSAIDs in the discovery group, but none of these sites were statistically significant in our replication group.

Conclusions

We found no replicable methylation differences in blood related to NSAID usage. If NSAID use does effect blood DNA methylation patterns, differences are likely small.     

Human liver epigenetic alterations in non-alcoholic steatohepatitis are related to insulin action

Both genetic and lifestyle factors contribute to the risk of non-alcoholic steatohepatitis (NASH). Additionally, epigenetic modifications may also play a key role in the pathogenesis of NASH. We therefore investigated liver DNA methylation, as a marker for epigenetic alterations, in individuals with simple steatosis and NASH, and further tested if these alterations were associated with clinical phenotypes. Liver biopsies obtained from 95 obese individuals (age: 49.5 ± 7.7 years, BMI: 43 ± 5.7 kg/m², type 2 diabetes [T2D]: 35) as a wedge biopsy during a Roux-en-Y gastric bypass operation were investigated. Thirty-four individuals had a normal liver phenotype, 35 had simple steatosis, and 26 had NASH. Genome-wide DNA methylation pattern was analyzed using the Infinium HumanMethylation450 BeadChip. mRNA expression was analyzed from 42 individuals using the HumanHT-12 Expression BeadChip. We identified 1,292 CpG sites representing 677 unique genes differentially methylated in liver of individuals with NASH (q < 0.001), independently of T2D, age, sex, and BMI. Focusing on the top-ranking 30 and another 37 CpG sites mapped to genes enriched in pathways of metabolism (q = 0.0036) and cancer (q = 0.0001) all together, 59 NASH-associated CpG sites correlated with fasting insulin levels independently of age, fasting glucose, or T2D. From these, we identified 30 correlations between DNA methylation and mRNA expression, for example LDHB (r = ?0.45, P = 0.003). We demonstrated that NASH, more than simple steatosis, associates with differential DNA methylation in the human liver. These epigenetic alterations in NASH are linked with insulin metabolism.     

Human metastable epiallele candidates link to common disorders

Metastable epialleles (MEs) are mammalian genomic loci where epigenetic patterning occurs before gastrulation in a stochastic fashion leading to systematic interindividual variation within one species. Importantly, periconceptual nutritional influences may modulate the establishment of epigenetic changes, such as DNA methylation at MEs. Based on these characteristics, we exploited Infinium HumanMethylation450 BeadChip kits in a 2-tissue parallel screen on peripheral blood leukocyte and colonic mucosal DNA from 10 children without identifiable large intestinal disease. This approach led to the delineation of 1776 CpG sites meeting our criteria for MEs, which associated with 1013 genes. The list of ME candidates exhibited overlaps with recently identified human genes (including CYP2E1 and MGMT, where methylation has been associated with Parkinson disease and glioblastoma, respectively) in which perinatal DNA methylation levels where linked to maternal periconceptual nutrition. One hundred 18 (11.6%) of the ME candidates overlapped with genes where DNA methylation correlated (r > 0.871; p < 0.055) with expression in the colon mucosa of 5 independent control children. Genes involved in homophilic cell adhesion (including cadherin-associated genes) and developmental processes were significantly overrepresented in association with MEs. Additional filtering of gene expression-correlated MEs defined 35 genes, associated with 2 or more CpG sites within a 10 kb genomic region, fulfilling the ME criteria. DNA methylation changes at a number of these genes have been linked to various forms of human disease, including cancers, such as asthma and acute myeloid leukemia (ALOX12), gastric cancer (EBF3), breast cancer (NAV1), colon cancer and acute lymphoid leukemia (KCNK15), Wilms tumor (protocadherin gene cluster; PCDHAs) and colorectal cancer (TCERG1L), suggesting a potential etiologic role for MEs in tumorigenesis and underscoring the possible developmental origins of these malignancies. The presented compendium of ME candidates may accelerate our understanding of the epigenetic origins of common human disorders.     

Age‐dependent DNA methylation patterns on the Y chromosome in elderly males

The Y chromosome, a sex chromosome that only exists in males, has been ignored in traditional epigenetic association studies for multiple reasons. However, sex differences in aging‐related phenotypes and mortality could suggest a critical role of the sex chromosomes in the aging process. We obtained blood‐based DNA methylation data on the Y chromosome for 624 men from four cohorts and performed a chromosome‐wide epigenetic association analysis to detect Y‐linked CpGs differentially methylated over age and cross‐validated the significant CpGs in the four cohorts. We identified 40–219 significant CpG sites (false discovery rate <0.05) with >82% of them hypermethylated with increasing age, which is in strong contrast to the patterns reported on the autosomal chromosomes. Comparing the rate of change in the Y‐linked DNA methylation across cohorts that represent different age intervals revealed a trend of acceleration in DNA methylation with increasing age. The age‐dependent DNA methylation patterns on the Y chromosome were further examined for their association with all‐cause mortality with results suggesting that the predominant pattern of age‐related hypermethylation on the Y chromosome is associated with reduced risk of death.     

Integrative analysis identifies potential DNA methylation biomarkers for pan-cancer diagnosis and prognosis

DNA methylation status is closely associated with diverse diseases, and is generally more stable than gene expression, thus abnormal DNA methylation could be important biomarkers for tumor diagnosis, treatment and prognosis. However, the signatures regarding DNA methylation changes for pan-cancer diagnosis and prognosis are less explored. Here we systematically analyzed the genome-wide DNA methylation patterns in diverse TCGA cancers with machine learning. We identified seven CpG sites that could effectively discriminate tumor samples from adjacent normal tissue samples for 12 main cancers of TCGA (1216 samples, AUC > 0.99). Those seven potential diagnostic biomarkers were further validated in the other 9 different TCGA cancers and 4 independent datasets (AUC > 0.92). Three out of the seven CpG sites were correlated with cell division, DNA replication and cell cycle. We also identified 12 CpG sites that can effectively distinguish 26 different cancers (7605 samples), and the result was repeatable in independent datasets as well as two disparate tumors with metastases (micro-average AUC > 0.89). Furthermore, a series of potential signatures that could significantly predict the prognosis of tumor patients for 7 different cancer were identified via survival analysis (p-value < 1e-4). Collectively, DNA methylation patterns vary greatly between tumor and adjacent normal tissues, as well as among different types of cancers. Our identified signatures may aid the decision of clinical diagnosis and prognosis for pan-cancer and the potential cancer-specific biomarkers could be used to predict the primary site of metastatic breast and prostate cancers.     

Sex-specific effects of early life cadmium exposure on DNA methylation and implications for birth weight

Dietary cadmium exposure was recently found to alter DNA methylation in adults, but data on effects early in life are lacking. Our objective was to evaluate associations between prenatal cadmium exposure, DNA methylation and birth weight. In total 127 mother-child pairs from rural Bangladesh were studied. For comparison, we included 56 children at 4.5 y. Cadmium concentrations in mothers’ blood (gestational week 14) and children’s urine were measured by ICPMS. Global DNA methylation was analyzed by Infinium HumanMethylation450K BeadChip in cord blood and children’s blood. Maternal cadmium exposure was associated with cord blood DNA methylation (p-value < 10^–16). The association was markedly sex-specific. In boys, 96% of the top 500 CpG sites showed positive correlations (r_S-values > 0.50), whereas most associations in girls were inverse; only 29% were positive (r_S > 0.45). In girls we found overrepresentation of methylation changes in genes associated with organ development, morphology and mineralization of bone, whereas changes in boys were found in cell death-related genes. Several individual CpG sites that were positively associated with cadmium were inversely correlated with birth weight, although none statistically significant after correction for multiple comparisons. The associations were, however, fairly robust in multivariable-adjusted linear regression models. We identified CpG sites that were significantly associated with cadmium exposure in both newborns and 4.5-y-old children. In conclusion, cadmium exposure in early life appears to alter DNA methylation differently in girls and boys. This is consistent with previous findings of sex-specific cadmium toxicity. Cadmium-related changes in methylation were also related to lower birth weight.     

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their relationships with chicken neoplastic disease resistance and susceptibility are not yet defined. In the present study, we analyzed the complexity of the DNA methylation variations and DNA mutations in the first exon of three DNMTs genes over generations, tissues, and ages among chickens of two highly inbred White Leghorn lines, Marek's disease-resistant line 6(3) and -susceptible line 7(2), and six recombinant congenic strains (RCSs). Among them, tissue-specific methylation patterns of DNMT3a were disclosed in spleen, liver, and hypothalamus in lines 6(3) and 7(2). The methylation level of DNMT3b on four CpG sites was not significantly different among four tissues of the two lines. However, two line-specific DNA transition mutations, CpG-->TpG (Chr20:10203733 and 10203778), were discovered in line 7(2) compared to the line 6(3) and RCSs. The methylation contents of DNMT1 in blood cell showed significant epimutations in the first CpG site among the two inbred lines and the six RCSs (P<0.05). Age-specific methylation of DNMT1 was detected in comparisons between 15 month-old and 2 month-old chickens in both lines except in spleen samples from line 7(2). No DNA mutations were discovered on the studied regions of DNMT1 and DNMT3a among the two lines and the six RCSs. Moreover, we developed a novel method that can effectively test the significance of DNA methylation patterns consisting of continuous CpG sites. Taken together, these results highlight the potential of epigenetic alterations in DNMT1 and DNMT3a, as well as the DNA mutations in DNMT3b, as epigenetic and genetic factors to neoplastic diseases of chickens.     

DNA methylation profiling of the vascular tissues in the setting of atherosclerosis

Currently, the question of epigenetic mechanisms of gene regulation in the context of cardiovascular diseases is of considerable interest. Here, DNA methylation profiles of vascular tissues of atherosclerotic patients have been analyzed for the first time using the Infinium Human Methylation27 BeadChip microarray (Illumina, United States). As the result, within 286 genes, 314 CpG sites that varied significantly in the level of DNA methylation between the tissue samples of carotid (in the area of atherosclerotic plaques and nearby macroscopically intact tissues of the vascular wall) and mammary arteries, as well as saphenous veins have been identified. The most pronounced differences in the methylation level was registered for CpG sites of homeobox genes HOXA2 and HOXD4, as well as the imprinted MEST gene. In particular, these genes were found to be hypomethylated in the carotid atherosclerotic plaques compared to their methylation patterns in intact tissues of internal mammary arteries and saphenous veins.     

Intra-Monozygotic Twin Pair Discordance and Longitudinal Variation of Whole-Genome Scale DNA Methylation in Adults

Monozygotic twins share identical genomic DNA and are indistinguishable using conventional genetic markers. Increasing evidence indicates that monozygotic twins are epigenetically distinct, suggesting that a comparison between DNA methylation patterns might be useful to approach this forensic problem. However, the extent of epigenetic discordance between healthy adult monozygotic twins and the stability of CpG loci within the same individual over a short time span at the whole-genome scale are not well understood. Here, we used Infinium HumanMethylation450 Beadchips to compare DNA methylation profiles using blood collected from 10 pairs of monozygotic twins and 8 individuals sampled at 0, 3, 6, and 9 months. Using an effective and unbiased method for calling differentially methylated (DM) CpG sites, we showed that 0.087%–1.530% of the CpG sites exhibit differential methylation in monozygotic twin pairs. We further demonstrated that, on whole-genome level, there has been no significant epigenetic drift within the same individuals for up to 9 months, including one monozygotic twin pair. However, we did identify a subset of CpG sites that vary in DNA methylation over the 9-month period. The magnitude of the intra-pair or longitudinal methylation discordance of the CpG sites inside the CpG islands is greater than those outside the CpG islands. The CpG sites located on shores appear to be more suitable for distinguishing between MZ twins.